EpIVax specializes in the development of epitope-driven vaccines by screening whole genomes for candidate vaccine components. In this application, we describe a rapid approach to the development of a tularemia vaccine that could be for BioDefense. First, F. tularensis genes that encode transmembrane, heat shock and secreted proteins will be screened for immunodominant epitopes by computer-driven (EpiMatrix) analysis of the bacterial genome. Limited in vitro assays will be performed to confirm MHC binding and immunogenicity of representative epitopes. Second, selected epitopes will be aligned and reverse translated in a string-of-beads formation into a DNA construct. The string of beads epitope construct will then be cloned into DNA plasmids optimized for strong and sustained expression in vivo. Third, we will vaccinate HLA-transgenic mice with the DNA vaccine constructs. De novo immune responses to the DNA vaccine constructs will be evaluated using intracellular cytokine flow cytometry. Fourth, we develop a model of protective immunity to aerosolized F. tularensis. Initially we will use this model to benchmark the protective efficacy of the LVS vaccine for later comparison studies. EpiVax will be responsible for selecting the F. tularensis epitopes and managing the project. The TB/HIV Research Lab at Brown University (subcontractor) will develop the oligonucleotide multi-epitope DNA vaccine constructs consisting of strings of the selected epitopes and Dr. Stephen Gregory (subcontractor, Liver Research Center, Lifespan Hospital) will perform the vaccination studies. The final product of Phase I will be an evaluation of the immunogenicity of several prototype tularemia vaccine constructs in transgenic mice and a functional challenge model. In Phase II we would screen additional epitopes, perform F. tularensis challenge studies, build the final vaccine constructs, optimize the immunogenicity of the final vaccine constructs with adjuvants and delivery vehicles and address the safety, toxicity and immunogenicity of the constructs in human subjects.